Rapid detection of Salmonella Enteritidis and S. Typhimurium serotypes and characterization of flagellar variants using real-time PCR.

The wide diversity of Salmonella serotypes poses challenges for traditional serotyping methods, which are labor-intensive and unsuitable for high-throughput testing, particularly in food safety contexts. Hence, this study aimed to develop and validate a real-time PCR method for detecting Salmonella spp., identifying the major public health serotypes (S. Enteritidis and S. Typhimurium), and differentiating monophasic S. Typhimurium variants. The method demonstrated high specificity and sensitivity, with 100 % inclusivity and 96.8 % exclusivity. Novel primers targeting fljB-hin allowed effective differentiation of monophasic variants, while dual biomarkers (STM4492 and STM2755) improved S. Typhimurium detection accuracy. Compared to conventional serotyping, the method showed excellent concordance (κ = 1.00, p < 0.01) and reduced turnaround time to under 2 h without requiring post-PCR processing. The inclusion of short amplicons (<200 bp) ensures efficiency and compatibility with fast cycling protocols. This validated, open-access assay offers a reliable and cost-effective molecular alternative for food safety monitoring, especially in settings with limited infrastructure.